Construction of a lentiviral vector for the expression of siRNA. (A) The template DNA for p53 siRNA (p53si) was directionally inserted into the Bgl II and HindIII sites of pSUPER. A second ClaI restriction site (asterisk) was created by adaptor ligation. The complete p53si expression cassette was excised from the modified pSUPER as a ClaI fragment and inserted into the lentiviral vector pWPXL (dotted line). LTR indicates long terminal repeat; ψ, psi packaging signal; RRE, Ref-responsive element; cPPT, central polypurine tract; EF1-α, human elongation factor alpha; EGFP, enhanced green fluorescent protein; WPRE, posttranscriptional cis-acting regulatory element of the woodchuck hepatitis virus; and LTR/SIN, self-inactivating 3′ long terminal repeat. (B) Western analysis of p53 protein expression in 293T cells. Control indicates nontransduced 293T cells; p53si, 293T cells transduced with pWPXL-p53si. (C) Assessment of lentiviral infection efficiency. CD34⁺ cells infected with pWPXL (empty vector), pWPXL-control-si (control si), or pWPXL-p53si (p53si) were analyzed for EGFP expression by flow cytometry. Cutoff used for cell sorting of EGFP-positive cells is shown (horizontal lines). FSC indicates forward side scatter. The percentages of EGFP-positive cells are given.